Project Summary/Abstract. Biofilm formation in many bacterial species is a major contributor to both colonization of host tissues and establishment of disease and is often mediated by signaling molecules that allow bacteria to sense their population density, and respond with the appropriate transcriptional response, in a process referred to as quorum sensing. Gram-positive bacteria are known to use both linear and non-linear peptide structures to mediate quorum responses, and a hallmark of the biology of Enterococcus faecalis is to use small hydrophobic peptides, called pheromones to mediate conjugative genetic transfer of plasmid between a donor and a recipient. We recently discovered an important ABC transporter that plays an important role in peptide secretion, the deletion of which significantly reduces conjugative mating efficiencies. Furthermore, the secreted peptide pheromones are known to act by being brought back into the cell through an oligopeptide permease complex, where the peptide binds a plasmid-encoded transcriptional repressor (PrgX) that results in derepression of conjugative functions. We found that both the ABC transporter and the oligopeptide permeases also significantly contributed to biofilm formation, which suggested that peptide signaling in E. faecalis also plays a major role in driving biofilm maturation. A family of transcription factors referred to as the RNPP family is comprised primarily of transcription factors known to bind small (5-8 amino acid) linear peptides and a query of the E. faecalis V583 genome, which includes two pheromone responsive plasmids (pTEF1 and pTEF2) identified several RNPP homologs, including the well-characterized PrgX-like protein involved in regulating conjugation responses by donor cells in E. faecalis. Additionally, we identified several chromosomally encoded RNPP homologs, including EF0073 and EF1316. Deletion of the ef0073 gene resulted in increased biofilm biomass, suggesting that EF0073 may be serving to repress biofilm processes. Furthermore, a deletion of ef1316 resulted in a significant decrease in biofilm, suggesting that this RNPP family member might be required to activate gene expression related to biofilm development. We hypothesize that these two RNPP transcription factors likely regulate important aspects of biofilm development, and we will use RNA-Seq to identify the regulons governed by these two proteins. As enterococci are a leading cause of hospital-associated urinary tract infections and account for over 200,000 infections annually in the U.S., we propose to determine if biofilm processes contribute to UTI pathogenesis. We will examine these transcription factor mutants in a catheter implant model of murine urinary tract infection. Gaining insight into how signaling governed by RNPP family transcription factors contributes to biofilm development in this organism will provide us additional targets to pursue in developing novel strategies for treating an organism that has become increasingly refractory to conventional antibiotic therapies.